Field of the Invention
The present invention relates generally to the field of induced pluripotent stem (iPS) cells and more specifically to methods for promoting cell programming and iPS cell generation, particularly by modulating barrier pathways in somatic cell reprogramming.
Background Information
Generation of induced pluripotent stem cells (iPSCs) by ectopic expression of four transcription factors, Oct4, Sox2, Klf4, and cMyc, has generated enthusiasm in regenerative medicine and developmental biology. In human and mouse somatic cells, other than these four factor combination, iPSCs, which exhibit properties similar to embryonic stem (ES) cells, can be generated with an alternative set of four factors, namely, Oct4, Nanog, Lin28, and Sox2. A number of cell types from different tissues have been successfully reprogrammed. A major roadblock in iPSC derivation and therapeutic use is low reprogramming efficiency, typically from 0.01% to 0.2%. Previous efforts have focused on screening for small molecules to enhance reprogramming efficiency and on developing new methods for iPSC derivation. In addition, synthetic modified RNA- and miRNA-based strategies have been developed to enhance iPSC efficiency and the understanding of the mechanisms of IPSC generation.
Since the discovery of techniques to create cells closely resembling embryonic stem cells, various types of mouse and human somatic cells have been reprogrammed to establish iPSCs. These cells have acquired full capacity to differentiate into different lineages. Resultant differentiated cells reportedly function in vitro and in vivo and serve to correct various diseases in mouse models. Moreover, iPSCs have been generated from tissues of patients with different disease conditions and could be a valuable source to study those pathologies or for drug screening in vitro. Nonetheless, the reprogramming process suffers from extreme low efficiency.
In addition to being a landmark technological advance, the process of inducing pluripotency from differentiated cells also raises fundamental questions about the dynamics of epigenetic stability and its relationship to the potential of pluripotency of a given differentiated state. Several promising approaches have been employed to improve reprogramming efficiency and to address mechanisms of iPSC production. Small molecule-based methods have been employed based on the observation that the treatment of cells with DNA methyltransferase 1 (Dnmt1) inhibitors accelerates reprogramming. TGFβ inhibition also leads to more efficient iPSC induction, as does omission of Sox2 and cMyc. Interestingly, partially reprogrammed iPSCs can be created and then converted to become fully reprogrammed following treatment with factors such as methyl transferase inhibitors.
Genome-wide analysis of promoter binding and induction of gene expression by the four reprogramming factors demonstrates that they bind to similar targets in iPSCs and mouse embryonic stem (mES) cells and likely regulate similar sets of genes, and also shows that targeting of reprogramming factors is altered in partial iPSCs. Several groups reported that p53-mediated tumor suppressor pathways may antagonize iPSC induction. Both p53 and its downstream effector p21 are induced during reprogramming, and lowering the expression of both enhances iPSC colony formation. Since these proteins are up-regulated in most cells expressing the four reprogramming factors, and cMyc reportedly blocks p21 expression, it is unclear how forced expression of these four factors overcomes the cellular responses to oncogenes/transgenes overexpression and why only a very small population of cells becomes fully reprogrammed. By combined dual inhibition (2i) of mitogen-activated protein kinase signaling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukemia inhibitory factor (LIF), it has been demonstrated that somatic cell state influences the requirements for reprogramming; this raises the intriguing possibility of capturing pre-pluripotent cells that can later advance to ground state pluripotency. Although major advances in the iPSC field including mechanisms and unsolved issues have been recently reviewed, barrier pathways in somatic cell reprogramming are still largely unknown.
Currently, there is a need to both better understand molecular mechanisms underlying reprogramming and develop more efficient methods to generate iPSCs. Elegant approaches have been applied to identify pathways regulating reprogramming. For example, mRNA profiling of somatic cells, iPSCs generated from those cells and intermediate populations that emerge during reprogramming indicates that cells can become “trapped” in a partially reprogrammed state and that treatment with DNA methyl transferase inhibitors enables them to become fully reprogrammed. Genome-wide analysis of promoter binding of specific transcription factors supports the idea that DNA-binding and gene activation are altered in partially reprogrammed iPSCs. Moreover, several groups have shown that p53 pathways, which are activated following overexpression of oncogenic reprogramming factors, act as a major reprogramming barrier. Recent studies show that TGFβ signaling also inhibits reprogramming and perturbs the mesenchymal-to-epithelial transition (MET), a process that enhances reprogramming and is regulated by microRNAs. However, there remains little information about how terminally differentiated cells are reprogrammed to an ES-like state by four transcriptional factors.
Much effort has gone into identifying factors that enhance iPSC derivation. In addition to small molecules that can reportedly replace some reprogramming factors, some compounds are known to enhance overall reprogramming efficiency in the presence of the classic four factors (4F), namely, Tgfbr inhibitors, AZA, vitamin C and VPA. Although some investigators report that VPA treatment dramatically enhances iPSC generation, more recent reports have reexamined the effects of the compound and found them to be modest. Therefore, currently only a limited number of compounds are available to enhance iPSC generation.
Kinases promote phosphorylation of targets by transferring phosphate groups from high-energy donors such as ATP. Kinases regulate many key processes such as cell cycle events and metabolic switching. However, few kinases have been shown to function in the reprogramming process. Given their critical function in numerous signaling pathways, unidentified kinases may modulate the reprogramming process. Additionally, iPSC generation might be significantly enhanced by manipulating their activity.